Polymyxin Stereochemistry and Its Role in Antibacterial Activity and Outer Membrane Disruption

With increasing rates of resistance toward commonly used antibiotics, especially among Gram-negative bacteria, there is renewed interested in polymyxins. Polymyxins are lipopeptide antibiotics with potent anti-Gram-negative activity and are generally believed to target lipid A, the lipopolysaccharide (LPS) anchor found in the outer membrane of Gram-negative bacteria. To characterize the stereochemical aspects of their mechanism(s) of action, we synthesized the full enantiomers of polymyxin B and the polymyxin B nonapeptide (PMBN). Both compounds were compared with the natural compounds in biological and biophysical assays, revealing strongly reduced antibacterial activity for the enantiomeric species. The enantiomeric compounds also exhibit reduced LPS binding, lower outer membrane (OM) permeabilization, and loss of synergetic potential. These findings provide new insights into the stereochemical requirements underlying the mechanisms of action of polymyxin B and PMBN

A β-Hairpin Epitope as Novel Structural Requirement for Protein Arginine Rhamnosylation

Protein N-glycosylation is ubiquitously present in all domains of life, and confers a plethora of functions to the protein including increased solubility, protection from degradation, interaction with receptors, and activation for function. For canonical asparagine glycosylation, the recognition sequence that directs glycosylation at specific asparagine residues is well-established. It generally holds for protein glycosylation that the primary amino acid sequence is most important for substrate recognition. Here we reveal that a recently discovered bacterial enzyme called EarP, that transfers rhamnose to a specific arginine residue in its acceptor protein EF-P, specifically recognizes a β-hairpin loop. Notably, while the rhamnosyltransferase activity of EarP is abolished when presented with linear substrate peptide sequences derived from EF-P in vitro, the enzyme readily glycosylates the same sequence when presented in a cyclized β-hairpin mimic containing an l-Pro-d-Pro motif. Additional studies with other substrate-mimicking cyclic peptides revealed that EarP activity is sensitive to the method used to induce cyclization and in some cases is tolerant to amino acid sequence variation. Using detailed NMR approaches, we established that the active peptide substrates all share some degree of β-hairpin formation, and therefore conclude that the β-hairpin epitope is the major determinant of arginine-rhamnosylation by EarP. Our findings add a novel recognition motif to the existing knowledge on substrate specificity of protein glycosylation, and are expected to inform future identifications of rhamnosylation sites in other protein substrates.</p

Chemical Proteomics Reveals Antibiotic Targets of Oxadiazolones in MRSA

Phenotypic screening is a powerful approach to identify novel antibiotics against methicillin-resistant Staphylococcus aureus (MRSA) infection, but elucidation of the targets responsible for antimicrobial activity is often challenging in the case of compounds with a polypharmacological mode-of-action. Here, we show that activity-based protein profiling maps the target interaction landscape of a series of 1,3,4-oxadiazole-3-ones, identified in a phenotypic screen to have high antibacterial potency against multidrug resistant S. aureus. In situ competitive and comparative chemical proteomics with a tailor-made activity-based probe, in combination with transposon and resistance studies, revealed several cysteine and serine hydrolases as relevant targets. Our data showcase oxadiazolones as novel antibacterial chemotype with a polypharmacological mode-of-action, in which FabH, FphC and AdhE play a central role

Dual targeting of the class V lanthipeptide antibiotic cacaoidin

Summary: Antibiotic resistance is reaching alarming levels, demanding for the discovery and development of antibiotics with novel chemistry and mechanisms of action. The recently discovered antibiotic cacaoidin combines the characteristic lanthionine residue of lanthipeptides and the linaridin-specific N-terminal dimethylation in an unprecedented N-dimethyl lanthionine ring, being therefore designated as the first class V lanthipeptide (lanthidin). Further notable features include the high D-amino acid content and a unique disaccharide substitution attached to the tyrosine residue. Cacaoidin shows antimicrobial activity against gram-positive pathogens and was shown to interfere with peptidoglycan biosynthesis. Initial investigations indicated an interaction with the peptidoglycan precursor lipid IIPGN as described for several lanthipeptides. Using a combination of biochemical and molecular interaction studies we provide evidence that cacaoidin is the first natural product demonstrated to exhibit a dual mode of action combining binding to lipid IIPGN and direct inhibition of cell wall transglycosylases

Dissecting the binding interactions of teixobactin with the bacterial cell wall precursor lipid II

The prevalence of life‐threatening, drug‐resistant microbial infections has challenged researchers to consider alternatives to currently available antibiotics. Teixobactin is a recently discovered “resistance‐proof” antimicrobial peptide that targets the bacterial cell wall precursor lipid II. In doing so, teixobactin exhibits potent antimicrobial activity against a wide range of Gram‐positive organisms. Herein we demonstrate that teixobactin and several structural analogues are capable of binding lipid II from both Gram‐positive and Gram‐negative bacteria. Furthermore, we show that when combined with known outer membrane‐disrupting peptides, teixobactin is active against Gram‐negative organisms.</p